Department of Animal Ecology, Ruhr-University Bochum, D-44780 Bochum, Germany.
BMC Mol Biol. 2010 Jun 29;11:50. doi: 10.1186/1471-2199-11-50.
The planktonic microcrustacean Daphnia pulex is among the best-studied animals in ecological, toxicological and evolutionary research. One aspect that has sustained interest in the study system is the ability of D. pulex to develop inducible defence structures when exposed to predators, such as the phantom midge larvae Chaoborus. The available draft genome sequence for D. pulex is accelerating research to identify genes that confer plastic phenotypes that are regularly cued by environmental stimuli. Yet for quantifying gene expression levels, no experimentally validated set of internal control genes exists for the accurate normalization of qRT-PCR data.
In this study, we tested six candidate reference genes for normalizing transcription levels of D. pulex genes; alpha tubulin (aTub), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box binding protein (Tbp) syntaxin 16 (Stx16), X-box binding protein 1 (Xbp1) and CAPON, a protein associated with the neuronal nitric oxide synthase, were selected on the basis of an earlier study and from microarray studies. One additional gene, a matrix metalloproteinase (MMP), was tested to validate its transcriptional response to Chaoborus, which was earlier observed in a microarray study. The transcription profiles of these seven genes were assessed by qRT-PCR from RNA of juvenile D. pulex that showed induced defences in comparison to untreated control animals. We tested the individual suitability of genes for expression normalization using the programs geNorm, NormFinder and BestKeeper. Intriguingly, Xbp1, Tbp, CAPON and Stx16 were selected as ideal reference genes. Analyses on the relative expression level using the software REST showed that both classical housekeeping candidate genes (aTub and GAPDH) were significantly downregulated, whereas the MMP gene was shown to be significantly upregulated, as predicted. aTub is a particularly ill suited reference gene because five copies are found in the D. pulex genome sequence. When applying aTub for expression normalization Xbp1 and Tbp are falsely reported as significantly upregulated.
Our results suggest that the genes Xbp1, Tbp, CAPON and Stx16 are suitable reference genes for accurate normalization in qRT-PCR studies using Chaoborus-induced D. pulex specimens. Furthermore, our study underscores the importance of verifying the expression stability of putative reference genes for normalization of expression levels.
浮游小型甲壳动物水蚤是生态、毒理学和进化研究中研究最多的动物之一。该研究系统中持续引起人们兴趣的一个方面是，水蚤在暴露于捕食者（如幻影摇蚊幼虫 Chaoborus）时能够形成诱导防御结构的能力。水蚤的可用基因组草案正在加速研究，以鉴定赋予可塑表型的基因，这些表型通常由环境刺激提示。然而，对于定量实时 PCR（qRT-PCR）数据的基因表达水平的定量，不存在经过实验验证的一组内部对照基因，用于准确标准化 qRT-PCR 数据。
在这项研究中，我们测试了 6 种候选参考基因，用于标准化水蚤基因的转录水平；微阵列研究中选择了微管蛋白-α（aTub）、甘油醛-3-磷酸脱氢酶（GAPDH）、TATA 框结合蛋白（Tbp）突触蛋白 16（Stx16）、X 框结合蛋白 1（Xbp1）和 CAPON，一种与神经元型一氧化氮合酶相关的蛋白。另外还测试了一种基质金属蛋白酶（MMP）基因，以验证其对 Chaoborus 的转录反应，这在早期的微阵列研究中已经观察到。通过 qRT-PCR 从表现出诱导防御的幼年水蚤 RNA 中评估这 7 种基因的转录谱，与未处理的对照动物相比。我们使用 geNorm、NormFinder 和 BestKeeper 程序评估了基因用于表达归一化的个体适用性。有趣的是，Xbp1、Tbp、CAPON 和 Stx16 被选为理想的参考基因。使用软件 REST 进行的相对表达水平分析表明，两种经典管家候选基因（aTub 和 GAPDH）均显著下调，而预测的 MMP 基因则显著上调。aTub 是一种特别不合适的参考基因，因为在水蚤基因组序列中发现了 5 个拷贝。当应用 aTub 进行表达归一化时，Xbp1 和 Tbp 被错误地报告为显著上调。
我们的结果表明，Xbp1、Tbp、CAPON 和 Stx16 基因是使用 Chaoborus 诱导的水蚤标本进行 qRT-PCR 研究中准确归一化的合适参考基因。此外，我们的研究强调了验证假定参考基因表达稳定性以归一化表达水平的重要性。
